Phosphorus stress-induced changes in plant root exudation could

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Environmental Processes

Phosphorus stress-induced changes in plant root exudation could potentially facilitate uranium mobilization from stable mineral forms. Nimisha Edayilam, Dawn A Montgomery, Brennan O Ferguson, Amith Sadananda Maroli, Nicole Martinez, Brian A. Powell, and Nishanth Tharayil Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b05836 • Publication Date (Web): 03 May 2018 Downloaded from http://pubs.acs.org on May 4, 2018

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Environmental Science & Technology

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Phosphorus stress-induced changes in plant root exudation could

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potentially facilitate uranium mobilization from stable mineral forms.

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Nimisha Edayilam†, Dawn Montgomery‡, Brennan Ferguson‡, Amith S. Maroli†, Nicole

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Martinez‡, Brian A. Powell‡, Nishanth Tharayil*†

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†Department of Plant & Environmental Sciences, Clemson University, Clemson, SC 29634, US;

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‡Department of Environmental Engineering and Earth Sciences, Clemson University, 342

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Computer Court, Anderson, SC 29625, USA.

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ABSTRACT

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Apparent deficiency of soil mineral nutrients often triggers specific physio-morphological

10

changes in plants, and some of these changes could also inadvertently increase the ability of

11

plants to mobilize radionuclides from stable mineral forms. This work, through a series of sand-

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culture, hydroponics and batch-equilibration experiments, investigated the differential ability of

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root exudates of Andropogon virginicus grown under conditions with variable phosphorus (P)

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availability (KH2PO4, FePO4, Ca3(PO4)2, and no P) to solubilize uranium (U) from the uranyl

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phosphate mineral Chernikovite. The mineral form of P, and hence the bioavailability of P,

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affected the overall composition of the root exudates. The lower bioavailable forms of P (FePO4

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and Ca3(PO4)2), but not the complete absence of P, resulted in a higher abundance of root

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metabolites with chelating capacity. With the lower P-bioavailability the physiological amino

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acid concentration inside of the roots increased, whereas the concentration of organic acids in the

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roots decreased due to the active exudation. In batch dissolution experiments, the organic acids,

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but not amino acids, increase the dissolution U from Chernikovite. The root exudate matrix of

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plants exposed to low available forms of P induced a >60% increase in U dissolution from

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Chernikovite due to 5-16 times greater abundance of organic acids in these treatments. However,

ACS Paragon Plus Environment


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this was ca. 70% of the theoretical dissolution achievable by this exudate matrix. These results

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highlight the potential of using active management of soil P as an effective tool to alter the plant-

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mediated mobilization of U in contaminated soil.

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INTRODUCTION

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Long-lived radionuclides released into the biosphere from the nuclear fuel cycle or processing of

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defense-related legacy wastes have the potential to lead to long-term subsurface soil

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contamination.1-2 The occurrence and fate of these long-lived radionuclides such as U in the

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environment is of growing concern because of the associated potential health and ecological

32

effects.

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concentration of the radionuclide source, the type and strength of the radiation being emitted, and

34

the mobility of radionuclides in soil.6-9 Due to adsorption and co-precipitation reactions,

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radionuclides in the soil are often present in chemical forms that are insoluble.10-11 Although

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these immobile forms are of limited environmental concern, various physical, chemical and

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biological process in soil could enhance the solubility and thus the mobility of these insoluble

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contaminants. The various process and the extent to which they influence the mobility of

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radionuclides are of key interest for environmental monitoring since the mobilized radionuclides

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pose a potential hazard.11 A finer level understanding of the processes that facilitate the mobility

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of radionuclides in the soil will not only assist in creating a predictive framework for

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radionuclide transport in the environment, but also will assist in formulating robust management

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practices that preclude the reactions that mobilize the radionuclide at the source.

3-5

The degree of hazard associated with radioactive contamination is determined by the

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The biochemical behavior of radionuclides in soils is correlated to its oxidation state.12,13

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Radionuclides such as Uranium (U), technetium (Tc), and plutonium (Pu) exhibit multiple

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oxidation states, and unlike the reduced forms that are insoluble, their oxidized forms are highly


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soluble.9.14 For instance, tetra and hexavalent states of U are dominant under environmental

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conditions with hexavalent, U(VI), U is the most stable state under oxidizing conditions and

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persists in solution as the dioxycation UO22+, whereas tetravalent U(IV) state hydrolyzes readily

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in water, precipitates as U(IV)-(hydr)oxide phases, and sorbs strongly to mineral surfaces. Thus,

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U(IV) is generally much less mobile than U(VI) in the environment.15 In addition to the

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oxidation state, the mobility of radionuclides in soil is also regulated by their sorption onto

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organic matter and mineral surfaces.16,17 Even though the chemical, physical, and geochemical

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processes that enhance the mobility of radionuclides in soils have been well studied across

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different model systems,12,18,19 relatively little is known about the plant-mediated processes that

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influence the mobility of radionuclides in soils.

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Potential mechanisms of plant-mediated transport of radionuclides in the soil can be

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broadly classified into the initial dissolution of radionuclides from the unavailable mineral forms

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(mobilization), followed by the uptake and accumulation of the mobilized radionuclides within

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the plant tissues,20 both of which are significantly influenced by the physiology of the plant. The

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plant uptake and transport of radionuclides in solution has been demonstrated in several lab-scale

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hydroponic studies.13,14 A few studies also have recorded significant upward movement of

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radionuclides in plants growing in contaminated soils.21-23 However, plant-mediated processes

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that regulate the mobilization of radionuclides at the source, and the influence of growing

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environments in modulating this plant-mediated radionuclide mobilization remains less explored.

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Soil nutrient deficiency is one of the key environmental stressors that limits growth and

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productivity of plants.24,25 Plants respond to the availability and distribution of nutrients in soils

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by modulating their root architecture in response to the resource availability.26-28 Along with the

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morphological changes, under nutrient deficiency plants also employ efficient physiological



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strategies to acquire nutrients from sources that are less soluble.29,30 At the physiological level,

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much research has focused on the components of root exudates that play important roles in

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increasing the bioavailability of nutrients.31 A common strategy across many plant species in the

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face of nutrient deficiency is rhizosphere acidification through proton extrusion.32 In addition,

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many plants exude organic acid anions and TCA cycle intermediates through root exudates that

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lower the rhizosphere pH. This lower soil pH potentially mobilizes the metal-bound P and

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enhances P acquisition capabilities of plants.32 Chelating compounds present in root exudates

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increase the mobilization and subsequent availability of nutrients because of their ability to form

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soluble complexes.33 Similarly, enhanced secretion of amino acids and organic acids have been

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reported under iron (Fe) deficiency.34,35 Recent experimental studies also have demonstrated the

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physiological capacity of plant roots to effectively remobilize mineral associated soil carbon.

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Root exudates released into soil respond to elevated CO2 concentration promotes removal of

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crosslinking metal cation from metal organic complex. 36 Root exudates have also been shown to

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alter the sorption/desorption dynamics of various contaminants. For instance, organic acids

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promoted desorption of phenanthrene and naphthalene has been reported recently.

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the above reactions including chelation and rhizosphere acidification could also influence the

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dissolution of radionuclides, especially from mineral forms where the radionuclides are

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complexed with nutrient elements that are essential for plant growth. Thus, nutrient deficiency

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could potentially influence the ability of plants to mobilize radionuclides. However, little is

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known regarding the role of nutrient stress in facilitating plant-mediated radionuclide

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mobilization in the soil. Many of the soils subjected to radionuclide contamination are marginal

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lands that challenge plant growth, and these sites are often dominated by ruderal plant species

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that are known for their ability to actively forage for nutrients39, a process that inadvertently


37,38

Many of

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could enhance the mobility of radionuclides from mineral forms that are otherwise unavailable.

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However, the ability of plants to mobilize radionuclides from stable mineral forms, and the

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environmental factors that regulate the magnitude of this mobilization remains less known.

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The current study focuses on the plant-induced mobilization of radionuclides under

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varying availability of P. Phosphorus is a key mineral required for plant growth, and unlike other

98

macronutrients, most P in soil is inaccessible to plants due to the pH-dependent complexation

99

with Ca, Fe and Al.29 To address this apparent deficiency, plants have evolved efficient

100

morphological and physiological strategies for obtaining this important macro-nutrient. These P

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foraging strategies include changes in root architecture, rhizosphere acidification and exudation

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of organic compounds with chelating moieties.29 As U is a ubiquitous (∼2−4 mg/kg of soil or

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sediment) naturally occurring radionuclide.40 Uranium has a strong affinity to associate with P,

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and is a major constituent in rock phosphate used for making P fertilizer.41 Also, injecting

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soluble P to soils for the sequestration of U as uranyl phosphate is an in situ remediation strategy

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that is widely practiced.42 Thus, under P limiting conditions the plant physiological adaptations

107

aimed at mobilizing P could potentially result in a collateral mobilization of U from uranyl

108

phosphates; but the magnitude and regulators of such mobilization remain unknown.

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In this work, a native bunch grass, Andropogon virginicus, exposed to different mineral

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forms of P was examined with respect to their potential for U mobilization. Andropogon

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virginicus is a ruderal, perennial grass species with an extensive deep root system (>2 m) that is

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tolerant to nutrient poor soil 43 and is one of the dominant ground covers in several radionuclide

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contaminated areas along Southeastern United States.44 The effect of P stress on U mobilization

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was determined using a series of sand, hydroponic culture, and batch dissolution studies. We

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hypothesized that, i) mineral forms of P will alter the root exudate profile of A. virginicus, and



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this change in exudate profile would be a function of the bioavailability of P; ii) root exudates of

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P deficient plants will be proportionally abundant in compound with a greater chelating capacity,

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and iii) root exudates of A. virginicus produced under P deficiency and will enhance the

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dissolution U from uranyl phosphate.

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MATERIALS AND METHODS

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1. Plant response under resource limitations

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1.1 Sand culture study

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The influence of bioavailability and spatial localization of P on root distribution patterns

124

and chemical composition of root exudates of A. virginicus was studied using sand culture study.

125

The experiment was conducted in 25 cm long, 5 cm diameter plastic tubes (PETG, Polyethylene

126

Terephthalate Glycol) filled with autoclaved sand (600 g per tube, 0.5 to 1mm grain size).

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Phosphorus treatments included three mineral forms with varying bioavailability of P: KH2PO4

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that represented a completely water-soluble form of P (Ksp = 28), and FePO4 and Ca3(PO4)2 that

129

are sparingly water soluble (Ksp = 1.3×10–22 and 2.0×10–29, respectively). The amount of the P

130

minerals across the three treatments were normalized to supply 36 mg P per 600 g of sand. Both

131

FePO4 and Ca3(PO4)2 were provided in two distinct spatial distribution patterns, minerals were

132

either uniformly distributed throughout the sand or concentrated in a 5 cm patch (Figure 1). In

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uniform distribution treatments, the tubes were first filled with pure autoclaved sand to a height

134

of 5 cm, and then topped with sand premixed with FePO4 or Ca3(PO4)2 to a height of 17 cm. For

135

patch treatments, the same amount of FePO4 or Ca3(PO4)2 as that in the uniform treatment was

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distributed in a 5 cm wide band 7 cm below the surface, resulting in a concentrated P distribution

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3.6 times that of the P in the uniform treatment (Figure 1). The control treatment contained pure

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autoclaved sand throughout the root zone, and KH2PO4 was provided along with Hoagland’s


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nutrient solution to form uniform distribution of P. Due to the high solubility of KH2PO4, patch

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application was not practical for this treatment. The background P concentration of the

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autoclaved sand was below the detection limit (0.1 mg P per kg sand).

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To investigate the role of the rhizosphere microbial community in aiding P mobilization,

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half the tubes in each of the two treatments (mineral form and spatial distribution of P) were re-

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inoculated with microbial inoculum native to the rhizosphere of A. virginicus. Details on

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rhizosphere soil collection and inoculum preparation is given in the supplementary information

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(SI Section S1). Four replicates were maintained for each of the 12 treatments (three forms of P

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minerals, two spatial distribution of P, and both autoclaved and re-inoculated soils). Seeds from

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A. virginicus, collected from Clemson, SC, were planted in a 16 x 8 tray containing a sterile

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germination mixture. Two-week-old seedlings were transplanted into these tubes and irrigated as

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needed with distilled water. Nutrient solutions specific to each treatment were supplied at weekly

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intervals: the control (KH2PO4) treatment received a complete nutrient solution providing 200

152

mg L-1 Ca as CaNO3, 234 mg L-1 K as KNO3 and KH2PO4, 30 mg L-1 P as KH2PO4, 48 mg L-1

153

Mg as MgSO4 ,140 mg L-1 N as NH4NO3, KNO3 and CaNO3 and micronutrients, 0.5 mg L-1 B as

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H3BO4, 4 mg L-1 Fe as FeSO4, 0.5 mg L-1 Zn as ZnSO4, 0.5 mg L-1 Mn as MnCl2, 0.02 mg L-1 Cu

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as CuSO4 , 0.01 mg L-1 Mo as (NH4)6MO7O. Chelating compounds including EDTA was

156

avoided from the micronutrient preparations.

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The calcium phosphate and iron phosphate treatments received a nutrient solution without P

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providing 200mg L-1 Ca as CaNO3, 195 mg L-1 as KNO3, 48mg L-1 Mg as MgSO4 and 140 mg

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L-1 N as NH4NO3 and KNO3. The plants were grown in a greenhouse maintained at 30/20 °C

160

day/night temperatures with a 14 h photoperiod for 16 weeks. The carbon assimilation capacity

161

of each plant was measured using Li-COR to compare the carbon assimilation rate across the



162

treatments. At the termination of experiment, the plants were harvested, sectioned into leaves and

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roots, and fresh biomass was recorded. The bulk sand was discarded and the sand adhering to the

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roots were gently removed and collected. Subsamples of root were stored in 70% ethanol for

165

determining the percent mycorrhizal colonization across different P treatments. Remaining root,

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shoot, and rhizosphere soils were rapidly frozen on dry ice and stored in a freezer at -80°C for

167

further analyses.

168

Extraction and analysis of metabolites

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Metabolites from the roots exposed to the P treatments were extracted using the method reported

170

in previous studies45,46 with minor modifications. Briefly, frozen root samples were finely ground

171

with dry ice using a mortar and pestle and stored at -800 C. Approximately, 1.0 g of the ground

172

samples were placed in a 15-mL centrifuge tube, and 4mL of ice-cold methanol: propanol

173

(1:1, v/v) was added to each. The tubes were vortexed for 20 s and then homogenized by

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sonication at 50% amplitude for 3 min and vortexed again for 20 s. This mixture was

175

centrifuged for 5 min at 2500 rpm and the supernatant was collected. A 200 µL aliquot of the

176

supernatant was transferred to a glass insert containing 20 µL of a mix of retention time lock d27-

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myristic acid (2 mg mL–1) and internal standard ribitol (500 µg mL–1) and dried completely under

178

nitrogen. Twenty µl of freshly prepared methoxylamine (20 mg mL–1) solution in pyridine was

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added, and the vials were incubated at 60°C for 90 min followed by trimethyl silylation with 90

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µL of N-methyl-N (trimethylsilyl) trifluoroacetamide (MSTFA) with 1% trimethylchlorosilane

181

(TMCS) for 30 min at 40 °C. These derivatized samples were then analyzed using a GC-MS

182

(Agilent 7980, Agilent Technologies, Santa Clara, CA). Details on GC-MS parameters and

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spectral identification is provided in the Supporting Information (SI, section S2).


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1.2 Hydroponic study

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To assess the U dissolution efficiency of root exudates of A. virginicus released under P

186

deficiency a hydroponic study was conducted using various mineral forms that differed in their P

187

availability. The exudates from the hydroponic study were further characterized to identify the

188

major compounds that could be responsible for the dissolution of uranyl- phosphate. Following

189

germination, each seedling was transplanted to 2 L glass jars containing half-strength Hoagland’s

190

solution that contained all macro and micro nutrients, but without any EDTA. The jars were

191

covered with aluminum foil to prevent light from interfering with root growth. The nutrient

192

solution was aerated continuously using an air pump and the nutrient solution was replaced with

193

fresh solution every three days to reduce the microbial buildup. The plants were grown in this

194

media for 40 days to facilitate the development of an extensive, healthy root system (SI Image

195

S1). Before the application of the P treatments, roots of the plants were gently but thoroughly

196

rinsed with deionized water multiple times, and all plants were kept in a P free, continuously

197

aerated, nutrient solution for 8 hours in dark to remove any traces of P that adhered to the roots.

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Plants of uniform visual characteristics (number of tillers, root length, and stem height) were

199

selected and randomly assigned to four treatment groups, P supplied as KH2PO4, Ca3(PO4)2,

200

FePO4, and no P, with five replicates per treatment. The plants were grown in their appropriate

201

modified Hoagland’s solution (SI section S3) for 72 hours with constant aeration; then 50 mL of

202

the solution in each flask was sampled using a syringe. Immediately after collection, the solution

203

was filter sterilized using 0.22-micron nylon membrane filters, and stored at -80℃ until analysis

204

of root exudates. Because of the co-occurring high salt content of the nutrient media, instead of

205

the MSTFA derivatization, the samples were subjected to ethyl chloroformate (ECF)

206

derivatization prior to GC-MS analysis to quantify the metabolites in the hydroponic solution.



207

The quantity of specific amino acids and organic acids was measured using external calibration

208

curves. Details on ECF derivatization method is provided in the Supporting Information (SI

209

section 4).

210

2. Effect of compounds identified in root exudates on the dissolution of uranyl-phosphate

211

Batch dissolution experiments were conducted to investigate the ability of various

212

compounds identified in the root exudates of A. virginicus to mobilize U from Chernikovite

213

(UO2HPO4•4H2O, lg Ksp; -24). A preliminary batch dissolution experiment was conducted using

214

citric acid,47 thereby optimizing (i) the amount of substrate (UO2HPO4•4H2O, (ii) the

215

concentration of the ligand and (iii) the substrate/solution ratio that would result in 50-60%

216

dissolution of the U from the UO2HPO4•4H2O. Based on the preliminary results (SI Figure S4)

217

0.5 mM of ligand, 25 mg uranyl-phosphate, and a 1:400 (0.0025g mL-1) solids/solution ratio was

218

used for the batch dissolution studies. Following optimization, batch dissolution experiments

219

were conducted on 13 compounds (ligands) identified as abundant in the root exudates of A.

220

virginicus. A background electrolyte solution of 0.01 M NaCl was used in the preparation of the

221

individual ligand stock solutions. Uranyl phosphate (0.025 g) was added to each of the 15 ml

222

centrifuge tubes, followed by 10 mL of the appropriate ligand solution. The control samples were

223

prepared in the same manner but with distilled water. The pH of the solution was then adjusted to

224

6 by adding small volumes of 0.1 M HCl or 0.1M NaOH. All samples were placed on an end-

225

over-end mixer for 24 hours at room temperature. After the mixing period, a 1.3 mL aliquot from

226

each tube was centrifuged at 8000 rpm for 20 min. The supernatant was filtered through a 0.22

227

µm syringe filter, and a 0.1 mL aliquot was diluted with 9 mL of 2% HNO3 for analysis via

228

inductively coupled plasma-mass spectrometry (ICP-MS, Thermo Scientific XSeries 2). The


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release of U from the uranyl-phosphate was expressed as the percentage of U released in relation

230

to the total U content in the uranyl phosphate.

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3. Root exudates under P deficiency after uranium dissolution

232

The ability of the overall root exudate matrix to mobilize U from uranyl-phosphate was

233

explored through a batch dissolution study similar to the one described previously. However,

234

instead of an individual ligand solution, we used hydroponic solutions exposed to various

235

nutrient treatments collected from Study 1.2. A batch-dissolution study was conducted using

236

uranyl-phosphate and collected root exudates in a solid/solution ratio 1:400; the pH of the

237

solution was adjusted to 6 by adding small volumes of 0.1 M HCl or 0.1 M NaOH. The samples

238

were incubated on a rotator for 24 hours. The control samples were prepared with fresh

239

Hoagland solution using the same process. The release of U from uranyl-phosphate was analyzed

240

as described above.

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Statistical analysis

242

A two-way analysis of variance (ANOVA) was used to compare the main and interactive effects

243

of inoculum (with and without inoculum) and nutrient treatments (KH2PO4, FePO4, Ca3(PO4)2,

244

and no P) on the biomass (root and shoot) and carbon assimilation capacity (photosynthesis)

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followed by Tukey’s HSD post hoc test. All differences were reported to be significant at P
amino acids. Although the

377

possibility of amino acids being involved in the chelation of metal ions has been discussed in

378

previous studies,61,71 observations from the current study indicate that, compared to organic

379

acids, amino acids have a lower effect on the release of U from uranyl-phosphate. The ligands

380

selected for this study contain COOH and NH2 groups as electron pair donor sites that attract

381

electron-deficient metals. The net charges of individual ligands vary depending on the degree of

382

dissociation of these functional groups, which is a function of the pH of the media.72 Most

383

chelating agents are less reactive at low pH,73 and pH range 5-6 generally leads to the

384

dissociation of the carboxyl groups, and as the pH increases further, the amino group are

385

deprotonated at pH 9-10.72 The organic acids used in this study carry 2-3 negatively charged

386

carboxyl groups, which form strong chelates with metals.72 Thus, at the pH used in this study (pH

387

6) the dissolution potential of organic acids was higher than the other ligands. In addition, types

388

and position of functional groups in the organic acids are most important in regulating metal

389

dissolution and subsequent leaching.73 For instance, comparing three dicarboxylic acids such as



390

malic acid, fumaric acid and succinic acid; malic acid was the most effective in mobilizing the U

391

from uranyl-phosphate due to the presence of alpha-hydroxyl group. These results emphasize the

392

importance of type of organic acids produced under P stress in dissolving/mobilizing U from

393

uranyl-phosphate in the soil.

394

3. Differential influence of root exudates produced under varying P-bioavailability on U

395

dissolution

396

Root exudates present a complex, multi-compound matrix, where individual compounds could

397

facilitate or antagonize the chelating capacity of the companion compounds. This, in turn, will

398

alter the chelation capacity of the overall exudate matrix. Batch dissolution studies were

399

conducted with the root exudates of A. virginicus exposed to different P treatments to determine

400

the differential ability of the overall exudate matrix to influence the dissolution of Chernikovite.

401

Root exudates from plant grown under no-phosphate and KH2PO4 had a lower effect on U

402

dissolution (20% dissolution). However, the root exudates collected from A. virginicus exposed

403

to less available forms of P treatments (FePO4) resulted in 70% dissolution of U from

404

Chernikovite (Figure 6b). The amount of U dissolution could be partly explained by the root

405

exudate composition- plants exposed to no-P and KH2PO4 treatments had a lower concentration

406

of organic acids whereas, the organic acids in the root exudates of plants exposed to FePO4

407

treatments were 5-16 times higher. The hydroponic study was not conducted in an axenic

408

condition since the presence of microbial cells and metabolites are required triggers to elicit

409

natural root exudation. Thus, the possibility that part of the observed mobilization could have

410

been from microbial exudates in the medium cannot be ruled out. The batch dissolution study

411

using the root exudates from hydroponic experiment, though instrumental in elucidating the

412

potential of multi-compound root exudates in mobilizing U from mineral forms, would


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overestimate the root exudate mobilization of U from soil matrices. This is because compounds

414

in soils the root exudates will be actively degraded by microbes, sorbed on to soil minerals, or

415

reabsorbed by plants, which in turn reduce the amount of chelating compounds in the

416

rhizosphere.

417

The percent dissolution of U from Chernikovite by the root exudates was 25 % lower than the

418

sum total of the dissolution potential of individual organic compounds (SI Figure S5). While the

419

concentration of individual organic compounds used for the batch dissolution study was

420

~0.5mM, total organic acid exuded under low P availability (FePO4) was 10 times greater (SI

421

Figure S3). Under P stress, chelation appears to be the major mechanism for P solubilization

422

form its metal-bound complexes (FePO4, Ca3(PO4)2), and thus would reduce the chelating

423

compounds that are available to complex with U. For instance, experimental results show that

424

presence of citric acid increases dissolution of U due to the formation of U(VI)-citrate

425

complexes. However, the formation of U(VI)-citric acid complexes could be regulated by the

426

dissolution of iron from FePO4 in the hydroponic solution as a consequence of the formation of

427

iron-citric acid complexes. Citrate can also form complexes with other micronutrients in the

428

hydroponic solution such as manganese, zinc, and copper.74 Thus, the presence of other metal

429

elements in the system can be one of the factors limiting the mobilization and potential leaching

430

of U in the soil. In addition, the natural exudates from plant roots are a complex mixture of

431

different class of biomolecules 37, and will vary substantially from the individual organic ligands

432

in function and character. Only small proportions compounds in the exudates, mostly low

433

molecular substances such as organic acids, sugars, amino acids, can be identified with a greater

434

molecular-level accuracy, whereas polymeric compounds such as proteins and carbohydrates are

435

often difficult to characterize. This has been shown previously for real and artificial root



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436

exudates (ARE), where ARE contained substantially higher DOC than plant root exudates.

437

Thus, there could be many more compounds in the root exudates of A. virginicus, some of which

438

could synergize or antagonize the overall dissolution of U form uranyl-phosphate, which would

439

have resulted in the observed lower mobilization of U from uranyl-phosphate by root exudates.

440

Although heavy metals dissolution as a function of root exudation has been studied before,74 our

441

study links the plant stress response to U mobilization via changes in the composition of root

442

exudates. Overall, the results indicate that when exposed to moderate nutrient stress, contributed

443

by low available forms of P, plants increase the amounts of chelating compounds in root

444

exudates to mobilize the sparingly available form of P in the soil. This, in turn, could potentially

445

facilitate the mobilization of U from uranyl-phosphate. Our results suggest that nutrient stress

446

could be one of the key factors that regulate plant-mediated mobilization of radionuclides in

447

soils. However, there are multiple fates for root exudates including microbial degradation, and

448

only a smaller fraction of the overall exuded compounds take part in nutrient foraging. Hence the

449

process of root exudate mediated mobilization of U would be slower under field conditions, but

450

could contribute significantly to the long-term soil transport of U at decadal time scale,

451

especially since the half-life of U could far exceed this time frame. Uranium is a key

452

contaminant of concern at several Department of Energy sites including the Savannah River Site

453

(SRS) in South Carolina. Additionally, soil injection of P is a remediation strategy that is

454

actively pursued across contaminated sites to decrease the mobility of U by forming a uranyl-

455

phosphate precipitate. Findings from this study suggest a possibility of enhanced plant-mediated

456

remobilization of U from the sequestered uranyl-phosphate, especially in soils with low P

457

availability. More importantly, though species-specific, the results from our study system

458

highlight the potential to use active soil P management as a viable strategy to regulate the plant-


Page 21 of 39


459

mediated U mobilization from contaminated soils. Although plant nutrient foraging processes

460

could inadvertently enhance the mobility of U from uranyl-phosphate, there are multiple fates

461

associated with mobilized U under natural environmental conditions. For example, once

462

mobilized the uptake of U from solution has been shown to occur across many plant species.75,76

463

However, if the mobilization of radionuclides exceeds their uptake then the radionuclides could

464

migrate down and potentially contaminate groundwater. Thus, future studies are still needed to

465

gain better insight into the relationship between soil P status in terms of plant bioavailability and

466

the associated U uptake and transport in plants and soil.

467

AUTHOR INFORMATION

468

Corresponding Author

469

*Phone: (864)656-4453 E-mail: [email protected]

470

Notes

471

The authors declare no competing financial interest.

472

ACKNOWLEDGEMENTS

473

We thank Dr. Vidya Suseela for assisting with the photosynthesis measurements and analysis of

474

root metabolomics data. This material is based upon work supported by the U.S. Department of

475

Energy Office of Science, Office of Basic Energy Sciences and Office of Biological and

476

Environmental Research under Award Number DE-SC-0001253, and DE-SC-0010832. This is

477

technical contribution No. ----- of the Clemson University Experiment Station.

478

Supporting Information.



479

Section S1: Preparation of soil inoculum, tissue phosphorus analysis. Section S2: GC parameters.

480

Section S3: Modified hoagland solution. Section S4: ECF derivatization. Image S1: Root image

481

from hydroponic culture study. Image S2: Mycorrhizal staining. Figure S1: Effect of phosphorus

482

treatment on root to shoot weight ratio. Figure S2: Abundance of organic acids in the rhizosphere

483

sand. Figure S3: Concentration of identified organic acids and amino acids in the root exudates

484

in hydroponic media. Figure S4: Dissolution of U form uranyl-phosphate using different

485

concentration of citric acid and uranyl-phosphate. Figure S5: Comparison between observed U

486

dissolution with root exudate matrix and expected dissolution with all individual ligands together

487

Reference

488

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687

FIGURES

688

689

(a)

(b)

690

691

692

693

694

695

Figure 1. Schematic of nutrient distribution used for the spatial localization of phosphorus. a)

696

Patch treatment b) Uniform treatment. Both treatments received the same amount of P.


Page 32 of 39

Page 33 of 39

697


(a)

698 699 700 701 702

703

704

705

(b) 706 707 708 709 710 711

(c)

712 713 714 715 716

717

Fig 2. Effect of different phosphorus (KH2PO4, FePO4, Ca3PO4) and reinoculation treatments on

718

(a) root biomass (b) shoot biomass and (c) photosynthesis of Andropogon virginicus grown in



719

sand culture. Different letters indicate significant difference between treatments (Tukey’s HSD

720

multiple comparison at P ≤ 0.05). Bars represent means ± SE (n = 4)

721

(a)

722 723 724 725 726 727

(b) 728 729 730 731 732 733 734

Fig.3 Concentration of phosphorus in the leaf (a) and root (b) of Andropogon virginicus grown

735

in sand culture exposed to P treatments (KH2PO4, FePO4 and Ca3(PO4)2). Different letters

736

indicate significant difference between treatments (Tukey’s HSD multiple comparison at P ≤

737

0.05). Bars represent means ± SE (n = 4)


Page 34 of 39

Page 35 of 39


(a)

738

739

740

741



742

(b)

743 744

Fig.4 Principle component analysis (PCA) and heat map showing changes in 73 metabolites in

745

Andropogon virginicus root tissue grown in sand culture exposed to P treatments (KH2PO4,

746

FePO4 and Ca3(PO4)2). a. PCA score plots of 73 metabolites. Data point represents biological

747

replicates, and the ellipses represent 95% confidence interval. b. Heat map and two-way

748

hierarchical cluster analysis of root metabolites highlighting the metabolite differences in

749

Andropogon virginicus in response to P treatments (KH2PO4, FePO4 and Ca3(PO4)2). The color


Page 36 of 39

Page 37 of 39


750

of each cell depicts the abundance of individual metabolites, blue indicates a significant decrease

751

and red represent a significant increase in metabolic content. 752 753 754 755 756 757 758 759 760 761 762 763 764 765

766

Fig.5 Average concentration of identified organic acids and amino acids in the root exudates of

767

Andropogon virginicus exposed to P treatments (KH2PO4, FePO4, Ca3(PO4)2 and no P) in

768

hydroponic media for 72 h. Bars represent means ± SE (n = 4).

769 770 771



772

Page 38 of 39

(a)

773 774 775 776 777 778 779 780 781 782 783 784

(b)

785 786 787 788 789 790 791 792

KH2PO4

No P

FePO4 Ca3(PO4)2

Blank

793

Fig 6. a. Uranium dissolution from uranyl-phosphate at pH 5 following treatment with different

794

solution matrix for 24 hours. a. 0.5 mM organic ligands; b. hydroponic solution from 72 hours of


Page 39 of 39


795

P treatments (KH2PO4, FePO4, Ca3(PO4)2 and No P). Different letters indicate significant

796

difference between treatments (Tukey’s HSD multiple comparison at P ≤ 0.05). Bars represent

797

means ± SE (n = 4)

798 799 800 801 802 803

Abstract figure

804


Phosphorus stress-induced changes in plant root exudation could

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